Further evidence of the in vivo role of erythropoietin or companion molecules induced by hypoxia on proliferation and continuing differentiation of BFU-e in PCDC

Normal and plethoric bone marrow cells were grown in plasma clot diffusion chambers (PCDC) implanted into the peritoneum of normal mice or mice submitted to 7 her of hypoxia (23,000 ft) daily, on a single day or on 2 consecutive days at different times after implantation of the PCDC's. Daily discontinuous hypoxia (DDH) produced more 6-day bursts than other treatments. Hypoxia on days 1 and 2 after implantation was nearly as effective as DDH on day-6 bursts. Later bouts of hypoxia or a singly hypoxic exposure on day 1 or 2 was less effective. Erythropoietin (Ep) levels were measured by bioassay on both diffusion chamber (DC) contents and serum. Serum Ep levels peaked at 160 mU/ml after a 7-hr hypoxic exposure while the DC content Ep levels were in the nondetectable range (less than 50 mU/ml). The data implies that either higher than normal Ep levels or a companion molecules (s) produced by hypoxia are required for 1-2 days early in the culture period of force an increasing number of BFU-d-e down the erythrocytic pathway and thus increase red cell production at times of need in vivo.     

Phospholipase abolishes the effect of stimulated platelets on the thrombin activation of factor VIII

Factor VIII functions as a cofactor in the intrinsic coagulation pathway and must first be activated to function optimally in this capacity. Low concentrations of thrombin activate factor VIII, and the presence of stimulated platelets is known to enhance the activation of factor VIII complexed to von Willebrand factor. The current studies show that platelets stimulated by thrombin, collagen, or calcium ionophore will increase the activation of isolated factor VIII by thrombin. Ongoing platelet release is not necessary for the enhanced factor VIII activation, nor is platelet von Willebrand factor or platelet membrane glycoproteins Ib or IIb/IIIa. Platelet membrane phospholipids, on the other hand, are important for the enhanced activation of factor VIII by thrombin because the effect of stimulated platelets is abolished by incubation of the stimulated platelets with phospholipases. These results suggest that the enhanced activation of factor VIII by thrombin in the presence of stimulated platelets may be mediated by factor VIII binding to platelet phospholipid or to a receptor whose functional integrity is dependent on surrounding membrane phospholipid.     

Association between morphologic distortion of sickle cells and deoxygenation-induced cation permeability increase

We hypothesized that the deoxygenation-induced increase in cation permeability of sickle cells was related to mechanical distention of the membrane by growing HbS polymer within the cell. To test this hypothesis, we determined the effect of deoxygenation on cation fluxes in sickle cells under conditions that restricted or permitted extensive growth of polymer, producing different degrees of membrane distention. Manipulation of suspending medium osmolality for density-isolated high and low mean cell hemoglobin concentration (MCHC) cells was used to regulate the extensional growth of polymer bundles and hence membrane distortion. For initially low MCHC cells, the deoxygenation-induced increase in both Na and K fluxes was markedly suppressed when the MCHC was increased by increasing the osmolality. This suppression corresponded to the inhibition of extensive morphologic cellular distortion. For initially high MCHC, ISC-rich cells, deoxygenation had minimal effect on K permeability. However, reduction of MCHC by a decrease in osmolality produced a concomitant increase in cation permeability and cellular distortion. These observations support the idea that the sickling-associated increase in membrane permeability is related to mechanical stress imposed on the membrane by bundles of HbS polymer.     

Paroxysmal nocturnal hemoglobinuria erythrocytes are of two distinct types: positive or negative for acetylcholinesterase

Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired disorder. Erythrocytes isolated from PNH patients show increased sensitivity to complement and decreased acetylcholinesterase (AChE) activity. In this study, indirect immunofluorescence analysis of a monoclonal antibody specific for a surface epitope of human erythrocyte AChE is used to quantitate the content of this enzyme at the single-cell level. Flow- cytofluorimetric analysis of erythrocytes from normal donors indicates that all erythrocytes contain detectable levels of the surface epitope with a strong correlation between cell size and enzyme content. In contrast, erythrocytes from PNH patients show two distinct populations of erythrocytes; namely, those containing a normal content of AChE and a second population containing no detectable AChE. The AChE-negative population of cells is quantitatively complement-sensitive. These data support suggestions that PNH is a clonal disorder resulting in two distinct types of circulating erythrocytes. The abnormal clone produces cells that are both surface-AChE-negative and complement-sensitive. In addition, the method described provides an attractive alternative for the diagnosis and quantitative evaluation of abnormal erythrocytes in PNH patients.     

Treatment of hypoplastic anemia in mice with placental transplants

A genetic mutation in mice (W/Wv) causes an autosomal recessive disease characterized by hypoplastic anemia which lasts throughout life. Double- dominant W/Wv anemic mice were sublethally irradiated to facilitate repopulation of marrow with transplanted cells and were injected intravenously with suspensions of 5-10 million placental cells of 15 days gestation derived from normal, isogeneic donors. Red cell counts fell promptly after irradiation and then rose progressively over a period of weeks, reaching normal levels of the nonmutant. Mean corpuscular volume and hemoglobin electrophoresis patterns of red cells in recipient W/Wv mice resembled those of normal donor animals. The therapeutic effect lasted for the duration of the observation period, in some instances over 9 mo. W/Wv mice that were administered Hanks' solution or fetal blood, instead of placental transplants, remained anemic. Late gestation placentas (18 days) were also ineffective.     

Peripheral blood harvest of unaffected CD34+ CD38- hematopoietic precursors in paroxysmal nocturnal hemoglobinuria

Paroxysmal nocturnal hemoglobinuria (PNH) arises from somatic mutation of a bone marrow progenitor that disrupts glycosylinositol phospholipid (GPI) anchoring of cell surface proteins. We recently characterized the expression of GPI-anchored decay acclerating factor (DAF) and CD59 during hematopoietic development in PNH marrow. We found that, although a subset of early hematopoietic precursors identified by the CD34+CD38- phenotype exhibits normal DAF and CD59 expression, DAF and CD59 are absent on the majority of CD34+CD38- cells. Pluripotent CD34+CD38- hematopoietic stem cells normally circulate in the peripheral blood and can be collected by apheresis, cryopreserved, and later used for reconstitution of hematopoiesis. In this study, we examined the phenotypes of CD34+ cells that are released into the blood of PNH patients. Analyses of apheresis samples from three affected individuals showed discrete populations of circulating DAF+CD59+CD34+ and DAF-CD59- CD34+ cells. Variable proportions of CD34+CD38- cells were present within the peripheral blood CD34+ cells of each patient, but in all three cases the DAF+CD59+CD34+CD38- cell subset subset. Because CD34+ cells lacking CD38 antigen are highly enriched for self-renewing hematopoietic stem cells, these findings indicate that apheresis samples can serve as a source of unaffected stem cells for autologous marrow transplantation of PNH patients.     

Structural integrity of the glycoprotein IIb and IIIa genes in Glanzmann thrombasthenia patients from Israel

Glanzmann thrombasthenia is an autosomal recessive disorder of the platelet glycoproteins (GP) IIb and IIIa. These glycoproteins normally serve as receptors for other adhesive glycoproteins, including fibrinogen, von Willebrand factor, and fibronectin. Most patients affected by Glanzmann thrombasthenia have low levels of GPIIb and GPIIIa; however, the separate mechanisms responsible for the deficiency in each remain to be determined. cDNA clones coding for the GPIIb and GPIIIa have been recently isolated, and their corresponding genomic sequences have been colocalized to the long arm of chromosome 17. Since a deletional event involving one or both of these structural genes could explain the disease phenotype, we have studied the DNA of two previously well-characterized cohorts of Glanzmann thrombasthenia patients from Israel. We performed Southern analysis with near full- length cDNA probes on genomic DNA obtained from 20 individuals. Four restriction enzyme digests were completed on each DNA sample. The similarity of banding patterns among probands, family members, and controls indicated that there were no major insertions or deletions in either the GPIIb or GPIIIa genes. Thus, the genetic defect in these patients with Glanzmann thrombasthenia is most likely due to either a small change in the nucleotide sequence of the coding region or a defect in the regulatory region of one or both genes.     

A clinical prediction model for long-term functional outcome after traumatic spinal cord injury based on acute clinical and imaging factors

Abstract To improve clinicians' ability to predict outcome after spinal cord injury (SCI) and to help classify patients within clinical trials, we have created a novel prediction model relating acute clinical and imaging information to functional outcome at 1 year. Data were obtained from two large prospective SCI datasets. Functional independence measure (FIM) motor score at 1 year follow-up was the primary outcome, and functional independence (score ≥6 for each FIM motor item) was the secondary outcome. A linear regression model was created with the primary outcome modeled relative to clinical and imaging predictors obtained within 3 days of injury. A logistic model was then created using the dichotomized secondary outcome and the same predictor variables. Model validation was performed using a bootstrap resampling procedure. Of 729 patients, 376 met the inclusion criteria. The mean FIM motor score at 1 year was 62.9 (±28.6). Better functional status was predicted by less severe initial American Spinal Injury Association (ASIA) Impairment Scale grade, and by an ASIA motor score >50 at admission. In contrast, older age and magnetic resonance imaging (MRI) signal characteristics consistent with spinal cord edema or hemorrhage predicted worse functional outcome. The linear model predicting FIM motor score demonstrated an R-square of 0.52 in the original dataset, and 0.52 (95% CI 0.52,0.53) across the 200 bootstraps. Functional independence was achieved by 148 patients (39.4%). For the logistic model, the area under the curve was 0.93 in the original dataset, and 0.92 (95% CI 0.92,0.93) across the bootstraps, indicating excellent predictive discrimination. These models will have important clinical impact to guide decision making and to counsel patients and families.     

Bone mineral density in partially recovered early onset anorexic patients - a follow-up investigation

Background and aims  

There still is a lack of prospective studies on bone mineral development in patients with a history of early onset Anorexia nervosa (AN). Therefore we assessed associations between bone mass accrual and clinical outcomes in a former clinical sample. In addition to an expected influence of regular physical activity and hormone replacement therapy, we explored correlations with nutritionally dependent hormones.